Escherichia coli is the most common cause of community acquired urinary tract infection (UTI) and a leading cause of nosocomial UTIs and sepsis. There are an estimated 8 million physician visits per year in the U.S for UTIs with significant associated morbidity and expense (> $1,000,000,000). We tested the hypothesis that virulence genes responsible for the pathogenesis of life-threatening E. coli extraintestinal infections, such as pyelonephritis and sepsis can be identified by comparison of the genome sequence of urosepsis E. coli strain CFT073 to either E. coli laboratory strain MG1655 or O157:H7 strain EDL933. We identified >300 CFT073-specific loci. The continued study of the region surrounding the D-serine deaminase genes (dsdCXA) is especially compelling. An allelic knockout mutant of dsdA, that encodes D-serine deaminase, is unaltered in expression of type 1 pili-mediated adherence, but 300-fold more competitive than the wild type strain in colonizing the bladder or kidney of mice infected in an ascending model of UTI. DsdC is a positive effector of dsdXA transcription and a member of the lysR-family of regulators. By in vivo and in vitro gene expression techniques we will test the hypotheses that D-serine through interaction with either dsdC or other co-effectors affects expression of multiple genes that directly influence CFT073 pathogenesis in murine models of disease. We will also identify environmental conditions and additional genes that affect the expression of the dsdCXA genes. Two such gene candidates are ipuAB (integrase-like proteins of uropathogens) that are immediately adjacent to the dsdCXA genes in the chromosome of CFT073 as well as other uropathogenic E. coli. ipuAB are homologs of the type 1 pili phase-switch recombinases, fimB and fimE that are linked to and control expression of the E. coli type 1 pilus fim operon. We will test the hypothesis that these genes provide an additional phase-switch system that controls expression of dsdCXA or other unknown genes. The objective of the proposed project is to identify and characterize critical virulence genes for E. coli involved in serious human diseases. This information will be of use for the development of new chemotherapeutic and vaccine strategies.